Transposon-Mediated Horizontal Transfer of the Host-Specific Virulence Protein ToxA between Three Fungal Wheat Pathogens

Abstract

Most known examples of horizontal gene transfer (HGT) between eukaryotes are ancient. These events are identified primarily using phylogenetic methods on coding regions alone. Only rarely are there examples of HGT where noncoding DNA is also reported. The gene encoding the wheat virulence protein ToxA and the surrounding 14 kb is one of these rare examples. ToxA has been horizontally transferred between three fungal wheat pathogens (Parastagonospora nodorum, Pyrenophora tritici-repentis, and Bipolaris sorokiniana) as part of a conserved ∼14 kb element which contains coding and noncoding regions. Here we used long-read sequencing to define the extent of HGT between these three fungal species. Construction of near-chromosomal-level assemblies enabled identification of terminal inverted repeats on either end of the 14 kb region, typical of a type II DNA transposon. This is the first description of ToxA with complete transposon features, which we call ToxhAT. In all three species, ToxhAT resides in a large (140-to-250 kb) transposon-rich genomic island which is absent in isolates that do not carry the gene (annotated here as toxa^- ). We demonstrate that the horizontal transfer of ToxhAT between P. tritici-repentis and P. nodorum occurred as part of a large (∼80 kb) HGT which is now undergoing extensive decay. In B. sorokiniana, in contrast, ToxhAT and its resident genomic island are mobile within the genome. Together, these data provide insight into the noncoding regions that facilitate HGT between eukaryotes and into the genomic processes which mask the extent of HGT between these species.IMPORTANCE This work dissects the tripartite horizontal transfer of ToxA, a gene that has a direct negative impact on global wheat yields. Defining the extent of horizontally transferred DNA is important because it can provide clues to the mechanisms that facilitate HGT. Our analysis of ToxA and its surrounding 14 kb suggests that this gene was horizontally transferred in two independent events, with one event likely facilitated by a type II DNA transposon. These horizontal transfer events are now in various processes of decay in each species due to the repeated insertion of new transposons and subsequent rounds of targeted mutation by a fungal genome defense mechanism known as repeat induced point mutation. This work highlights the role that HGT plays in the evolution of host adaptation in eukaryotic pathogens. It also increases the growing body of evidence indicating that transposons facilitate adaptive HGT events between fungi present in similar environments and hosts.

Keywords: ToxA; adaptive evolution; fungal pathogen; fungal wheat pathogen; horizontal transfer; transposon; wheat pathogen.

FIG 1

Characterization of ToxhAT in B. sorokiniana isolate CS10. (A) Self-alignment of ToxhAT drawn as a dot plot. The red line down the center indicates a 1-to-1 alignment; yellow lines show inverse alignments. Terminal inverted repeats (TIRs) and inverted repeats (IRs) are boxed. The TPRs are short tandem repeats found in the gene with the Patatin domain. (B) Alignment of 74-bp TIR1.1 and the reverse complement (RC) of TIR1.2. Gray bases indicates aligned positions that are identical between the two sequences. (C) Manual annotation of coding regions within ToxhAT showing each open reading frame (green), inverted repeat (blue), and TIR (light green).

FIG 2

(A) Overview of the ToxhAT in all three pathogen species. All features drawn to the right of the red dashed line are drawn to scale as indicated by the black scale bar at the bottom. Features to the left of the red dashed line are not drawn to scale; relative sizes are indicated with brackets. The opaque red rectangles drawn between the chromosomes outside the TIRs show regions of synteny as indicated by whole-chromosome alignment. The approximate percentage of nucleotide identity is indicated within the red shading. “(B)” in part A indicates the region shared between P. nodorum and P. tritici-repentis, which is drawn in part. (B) Closeup view of whole-chromosome alignment between P. nodorum and P. tritici-repentis. Chromosomes are drawn as thick black lines with positions of annotated transposons shown in colored blocks. Transposons are classified into superfamiles as indicated by the legend. The additional opaque red/blue boxes appearing above or below the chromosomes represent nucleotide regions that are >70% identical and were identified by whole-chromosome alignment with LASTZ. Black lines connect syntentic blocks aligned in the same direction, while blue lines connect inverted syntenic blocks. Trans, transposon.

FIG 3

Genomic context of the ToxhAT-containing region (red box) in each of the three species in comparison to toxa⁻ isolates. (A) Lastz alignment of the homologous chromosome between B. sorokiniana ToxA⁺ isolate CS10 and toxa⁻ isolate CS27. Blue blocks drawn on the chromosome maps (black lines) represent the location of annotated transposons within each genome. Red ribbons drawn between the two isolates represent syntenic alignments found in Lastz that showed more than 70% identity and were greater than 2 kb in length. Blue ribbons drawn between the two isolates show inversions between the two genomes. (B) Lastz alignment of the homologous chromosome between P. nodorum ToxA⁺ isolate SN15 and toxa⁻ isolate Sn79-1087. The coloring scheme is the same as that used in panel A. (C) Isolate Ptr1C-BFP with repeat regions shown in the blue blocks along the chromosome (black line). The average Illumina coverage for 10 kb windows across the chromosome is indicated at the bottom. The color of the line corresponds to the proportion of bases within the 10 kb window that had nonzero coverage.

FIG 4

ToxhAT within B. sorokiniana is mobile in two distinct ways. (A) Alignment of chromosomes 01 and 08 of CS10 against WAI2406 homologous chromosomes. The ToxhAT (red line/box drawn on outer circle) is located on Chr01 in WAI2406 along with a large region of repeat-rich DNA (black boxes in outer circle). (B) The corrected and trimmed WAI2406 Nanopore reads aligned to the de novo version of itself. The black dotted line shows a slope value of 1, which indicates that the read aligned base per base to the chromosome shown on the x axis. Reads with a slope value different from 1 are reads that have been mapped discontinuously (i.e., with large insertions or deletions). The blue blocks show the translocated DNA, found in chromosome 08 in CS10 but in chromosome 01 in WAI2406. The red box indicates the position of ToxhAT. (C) The data correspond to the same reads as those described for panel B but represent alignment to chromosome 08 of CS10. Note that the read alignment is not continuous and breaks at the translocation edges. (D) Alignment of chromosome 08 from CS10 against tig17 from WAI2411. (E) The data are presented as described for panel B but represent isolate WAI2411. The red block shows the position of ToxhAT. (F) The data are presented as described for panel C but represent isolate WAI2411. Note that no reads with a slope value of 1 extend beyond the ToxhAT itself (red box).